Regulation of Translation of Heterologously Expressed Genes

ABSTRACT

The present invention pertains to a method of expressing a protein of interest, preferably a heterologous protein, in preferably a plant. In a preferred embodiment said plant is a doubled haploid homozygous transgenic  Nicotiana tabacum  plant silenced for Ntp303. Furthermore, the invention relates to said plant with or without nucleic acid constructs according to the invention. Propagation, harvest and tissue material of said transgenic  Nicotiana tabacum  plant is also a part of the invention.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application claims priority to U.S. patent application Ser. No.10/491,749 filed on Apr. 5, 2004, which is national phase application ofInternational Application No. PCT/NL02/00638 filed on Oct. 4, 2002,which claims priority to U.S. patent application Ser. No. 60/327,003filed on Oct. 5, 2001, to EP 01203772.7 filed on Oct. 5, 2001, and to EP02076593.9 filed Apr. 19, 2002, all of which are hereby incorporatedherein by reference in their entireties.

BACKGROUND OF THE INVENTION

Sexual reproduction in plants and animals requires the production ofgametes. Although many cytological differences exist in thedevelopmental stages of gamete development between the plant and animalkingdoms, several parallels exist. In plants as well as in animals,gamete production is a highly ordered process characterised bytransitions of stem cells from one physiological state to the next bymitotic and meiotic divisions. These series of events are directed bymultiple changes in gene expression. Several stages in gametedevelopment in several plant and animal species proceed almost withoutany transcriptional activity. In these stages, previously synthesisedmRNAs are translated into products that are essential for furtherdevelopment. This implies that, in these species, post-transcriptionalcontrol of gene expression is the leading principle in gametedevelopment. An example of post-transcriptional regulation of geneexpression during gamete development is the maturation and germinationof the male gametophyte (pollen) in angiosperm plants. Immature pollenconsists of a small generative cell and a large vegetative cell whichare formed out of microspores through an asymmetric haploid mitoticdivision (pollen mitosis 1). During the subsequent stages in developmentsome species have a second haploid mitosis (pollen mitosis II) resultingin tricellular pollen, a process absent in most species (i.e. bicellularpollen). Maturation of this bi- and tricellular pollen is completed by arange of developmental processes which finally result in a progressivedehydration of the pollen and its transition to dormancy. Maturation ofpollen of several plant species is accompanied by an accumulation oflarge quantities of rRNAs, tRNAs, mRNAs and ribosomes. As soon as thepollen lands on a compatible stigma, an extensive rehydration of thepollen grain occurs leading to a rapid reactivation of the translationmachinery which uses the previously accumulated tRNAs, rRNAs and mRNAs.The proteins that are synthesized from these stored products arerequired for the progamic life stage of the pollen, i.e. germination ofthe pollen, the subsequent growth of the pollen tube and the secondhaploid mitosis in case of the bicellular pollen.

Despite the importance of post-transcriptional processes for theregulation of pollen gene expression, little is known about themechanisms underlying post-transcriptional regulation of pollen geneexpression. There is only one study in the art that focuses on thetranslational regulation of a pollen expressed gene (lat52) (Bate etal., (1996) Plant J., 10(4), 613-623). In this study, the involvement ofthe 5′ UTR in the pollen specific regulation of translation has beendemonstrated. More in general, the rate of translation is influenced bycis-acting elements in mRNAs. E.g. the translation level of mRNA speciesfrom different eukaryotic systems is modulated by cis-acting elements inthe 5′ UTR, the coding sequence, or the 3′ UTR. These cis-actingelements act by influencing mRNA stability, translation initiation orelongation.

The regulation of the synthesis of the tobacco pollen protein NTP303takes place at the post-transcriptional level. Transcripts of the ntp303gene are first detectable after pollen mitosis I and continue toaccumulate during pollen maturation and subsequent pollen tube growth(Weterings et al., (1992) Plant Mol. Bioi. 18(6), 1101-1111). Incontrast, the protein only appears in detectable amounts at the onset ofpollen rehydration (Wittink et al., (2000) Sex. Plant Reprod. 12(5),276-284). Thus, despite the accumulation of its mRNA there is noefficient synthesis of the NTP303 protein during pollen development,which constraint is only relieved at the onset of pollen germination. Itis, however, not clear what cis-acting elements in the npt303 mRNA, ifany, are responsible for this mechanism of translational regulation. Inparticular it is not clear whether any such elements could be used toregulate the expression of proteins other than NPT303, such asheterologous proteins.

DESCRIPTION OF THE FIGURES

FIG. 1: Schematic representation and names of the UTR gene fusionconstructs used in the present study.

FIG. 2: Transient expression of UTR gene fusion constructs in developingpollen (A) and growing pollen tubes (13). Rlu/10 sec⁻¹ means therelative light units per 10 seconds measuring time as determined bynormalization of the absolute expression of the test construct with thatof the reference construct.

FIG. 3: Transient expression of UTR gene fusion constructs in developingpollen (A) and growing pollen tubes (B). Rlu/10 sec⁻¹ means the relativelight units per 10 seconds measuring time as determined by normalizationof the absolute expression of the test construct with that of thereference construct.

FIG. 4: Transient expression of UTR gene fusion constructs containingthe Renilla luciferase coding region in growing pollen tubes. Rlu/10sec⁻¹ means the relative light units per 10 seconds measuring time asdetermined by normalization of the absolute expression of the testconstruct with that of the reference construct.

FIG. 5: Transient expression of UTR gene fusion constructs containingthe CaMV 35S promoter in growing pollen tubes (A) and young leaves (B3).Rlu/10 sec⁻¹ means the relative light units per 10 seconds measuringtime as determined by normalization of the absolute expression of thetest construct with that of the reference construct.

FIG. 6: Predicted secondary structure of the 5′ UTR of ntp303. Structureprediction and the calculation of the ΔG value was performed using theRNAdraw software package. H-I (nucleotides 4-76 of SEQ ID NO: 1) andH-II (nucleotides 104-151 of SEQ ID NO: 1) represents two predictedhairpin-loop structures. See result section for a description of the 5′UTR.

FIG. 7: Schematic representations and expression effects of ntp303 5′UTR H-I (A and B) and H-II (C and D) mutations. FIG. 7A shows thesequences for Δ29 303 5′/35s 3′ (SEQ ID NO: 15); Δ55 303 5′/35S 3′(SEQID NO: 16); and ΔGAA 303 5′/35S 3′ (SEQ ID NO: 17). FIG. 7B includes 3035′35S 3′(SEQ ID NO: 18). FIG. 7C shows the sequences for Δ70 303 5′/35s3′ (SEQ ID NO: 19) and ΔH-II 303 5′/35S 3′ (SEQ ID NO: 20). FIG. 7B andFIG. 7D reflect the effect of the constructs of FIG. 7A and FIG. 7C,respectively, upon the pollen tube growth. Rlu/10 sec.⁻¹ means therelative light units per 10 seconds measuring time as determined bynormalization of the absolute expression of the test construct with thatof the reference construct. Measurements were assayed after 20 hours ofpollen tube growth.

DESCRIPTION OF THE INVENTION Definitions

Herebelow follow definitions of terms as used in the invention.

Plant

As used herein, the term “plant” refers to either a whole plant,including in general the class of higher plants amenable totransformation techniques, including both monocotyledonous anddicotyledonous plants or a part of a plant such as e.g. roots, stems,stalks, leaves, petals, fruits, seeds, tubers, pollen, meristems,callus, sepals, bulbs and flowers. The term plant as used herein furtherrefers, without limitations, to plant cells in seeds, suspensioncultures, embryos, meristematic regions, callus tissue, leaves, roots,shoots, gametophytic and sporophytic tissue, pollen, protoplasts andmicrospores. Furthermore, all plant tissues in all organs are includedin the definition of the term plant as used herein. Plant tissuesinclude, but is not limited to, differentiated and undifferentiatedtissues of a plant, including pollen, pollen tubes, pollen grains,roots, shoots, shoot meristems, coleoptilar nodes, tassels, leaves,cotyledonous petals, ovules, tubers, seeds, kernels. Tissues of plantsmay be in planta, or in organ, tissue or cell culture. As used herein,monocotyledonous plant refers to a plant whose seeds have only onecotyledon, or organ of the embryo that stores and absorbs food. As usedherein, dicotyledonous plant refers to a plant whose seeds have twocotyledons. Plants included in the invention are all plants amenable totransformation.

Operably Linked

As used herein, the term “operably linked” refers to two or more nucleicacid sequence elements that are physically linked and are in afunctional relationship with each other. For instance, a promoter isoperably linked to a coding sequence if the promoter is able to initiateor regulate the transcription or expression of a coding sequence, inwhich case the coding sequence should be understood as being “under thecontrol of” the promoter. Generally, when two nucleic acid sequences areoperably linked, they will be in the same orientation and usually alsoin the same reading frame. They usually will be essentially contiguous,although this may not be required.

Promoter

As used herein, the term “promoter” refers to a nucleic acid fragmentthat functions to control the transcription of one or more genes,located upstream with respect to the direction of transcription of thetranscription initiation site of the gene, and is structurallyidentified by the presence of a binding site for DNA-dependent RNApolymerase, transcription initiation sites and any other DNA sequences,including, but not limited to transcription factor binding sites,repressor and activator protein binding sites, and any other sequencesof nucleotides known to one skilled in the art to act directly orindirectly to regulate the amount of transcription from the promoter.

Hybridising Nucleic Acid Orthologs and Hybridising 5′ UTRs

Any nucleotide sequence capable to hybridise to the nucleotide sequencesof SEQ ID NO. 1 is defined as being part of the 5′ UTR of the invention.Stringent hybridisation conditions are herein defined as conditions thatallow a nucleic acid sequence of at least 25, preferably 50, 75 or 100,and most preferably 150 or more nucleotides, to hybridise at atemperature of about 65° C. in a solution comprising about 1 M salt,preferably 6×SSC or any other solution having a comparable ionicstrength, and washing at 65° C. in a solution comprising about 0.1 Msalt, or less, preferably 0.2×SSC or any other solution having acomparable ionic strength. Preferably, the hybridisation is performedovernight, i.e. at least for 10 hours and preferably washing isperformed for at least one hour with at least two changes of the washingsolution. These conditions will usually allow the specific hybridisationof sequences having about 90% or more sequence identity. Moderatehybridization conditions are herein defined as conditions that allow anucleic acid sequence of at least 50, preferably 150 or morenucleotides, to hybridise at a temperature of about 45° C. in a solutioncomprising about 1 M salt, preferably 6×SSC or any other solution havinga comparable ionic strength, and washing at room temperature in asolution comprising about 1 M salt, preferably 6×SSC or any othersolution having a comparable ionic strength. Preferably, thehybridisation is performed overnight, i.e. at least for 10 hours, andpreferably washing is performed for at least one hour with at least twochanges of the washing solution. These conditions will usually allow thespecific hybridisation of sequences having up to 50% sequence identity.The person skilled in the art will be able to modify these hybridisationconditions in order to specifically identify sequences varying inidentity between 50% and 90%.

Homologous

The term “homologous” when used to indicate the relation between a given(recombinant) nucleic acid or polypeptide molecule and a given hostorganism or host cell, is understood to mean that in nature the nucleicacid or polypeptide molecule is produced by a host cell or organisms ofthe same species, preferably of the same variety or strain. Ifhomologous to a host cell, a nucleic acid sequence encoding apolypeptide will typically be operably linked to another promotersequence or, if applicable, another secretory signal sequence and/orterminator sequence than in its natural environment.

When used to indicate the relatedness of two nucleic acid sequences theterm “homologous” means that one single-stranded nucleic acid sequencemay hybridise to a complementary single-stranded nucleic acid sequence.The degree of hybridisation may depend on a number of factors includingthe extent of identity between the sequences and the hybridisationconditions such as temperature and salt concentration as discussedlater. Preferably the region of identity is greater than 5 bp, morepreferably the region of identity is greater than 10 bp.

Heterologous

The term “heterologous” when used with respect to a nucleic acid orpolypeptide molecule refers to a nucleic acid or polypeptide from aforeign cell which does not occur naturally as part of the organism,cell, genome or DNA or RNA sequence in which it is present, or which isfound in a cell or location or locations in the genome or DNA or RNAsequence that differ from that in which it is found in nature.Heterologous nucleic acids or proteins are not endogenous to the cellinto which they are introduced, but have been obtained from another cellor synthetically or recombinantly produced. Generally, though notnecessarily, such nucleic acids encode proteins that are not normallyproduced by the cell in which the DNA is transcribed or expressed,similarly exogenous RNA codes for proteins not normally expressed in thecell in which the exogenous RNA is present. Furthermore, it is knownthat a heterologous protein or polypeptide can be composed of homologouselements arranged in an order and/or orientation not normally found inthe host organism, tissue or cell thereof in which it is transferred,i.e. the nucleotide sequence encoding said protein or polypeptideoriginates from the same species but is substantially modified from itsnative form in composition and/or genomic locus by deliberate humanintervention. Heterologous nucleic acids and proteins may also bereferred to as foreign nucleic acids or proteins. Any nucleic acid orprotein that one of skill in the art would recognise as heterologous orforeign to the cell in which it is expressed is herein encompassed bythe term heterologous nucleic acid or protein. The term heterologousalso applies to non-natural combinations of nucleic acid or amino acidsequences, i.e. combinations where at least two of the combinedsequences are foreign with respect to each other.

Sequence Identity

“Sequence identity”, as known in the art, is a relationship between twoor more amino acid (polypeptide or protein) sequences or two or morenucleic acid (polynucleotide) sequences, as determined by comparing thesequences. In the art, the percentage of “identity” indicates the degreeof sequence relatedness between amino acid or nucleic acid sequences asdetermined by the match between strings of such sequences. Two aminoacid sequences are considered “similar” if the polypeptides only differin conserved amino acid substitutions. In determining the degree ofamino acid similarity, the skilled person takes into account“conservative” amino acid substitutions. Conservative amino acidsubstitutions refer to the interchange of amino acids having similarside chains. For example, a group of amino acids having aliphatic sidechains is glycine, alanine, valine, leucine, and isoleucine; a group ofamino acids having aliphatic-hydroxyl side chains is serine andthreonine; a group of amino acids having amide-containing side chains isasparagine and glutamine; a group of amino acids having aromatic sidechains is phenylalanine, tyrosine, and tryptophan; a group of aminoacids having basic side chains is lysine, arginine, and histidine; and agroup of amino acids having sulphur-containing side chains is cysteineand methionine. Preferred conservative amino acids substitution groupsare: valine-leucine-isoleuci-ne, phenylalanine-tyrosine,lysine-arginine, alanine-valine, and asparagine-glutamine.Substitutional variants of the amino acid sequence disclosed herein arethose in which at least one residue in the disclosed sequences has beenremoved and a different residue inserted in its place. Preferably, theamino acid change is conservative. Preferred conservative substitutionsfor each of the naturally occurring amino acids are as follows: Ala toser; Arg to lys; Asn to gln or his; Asp to glu; Cys to ser or ala; Glnto asn; Glu to asp; Gly to pro; His to asn or gln; Ile to leu or val;Leu to ile or val; Lys to arg; Asn to gln or glu; Met to leu or ile; Pheto met, leu or tyr; Ser to thr; Thr to ser; Trp to tyr; Tyr to trp orphe; and, Val to ile or leu.

“Identity” and “similarity” can be readily calculated by known methods,including but not limited to those described in Computational MolecularBiology, Lesk, A. M., ed., Oxford University Press, New York, 1988;Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York, 1993; Computer Analysis of Sequence Data, PartI, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heine, G., AcademicPress, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux,J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman,D., SIAM J. Applied Math., 48:1073 (1988).

Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Preferred computer program methods to determine identity and similaritybetween two sequences include e.g. the GCG program package (Devereux,J., et al., Nucleic Acids Research 12 (1):387 (1984)), BestFit and PASTA(Altschul, S. F. et al., J. Mol. Biol. 215:403-410 (1990). The BLAST 2.0family of programs which can be used for database similarity searchesincludes: BLASTN for nucleotide query sequences against nucleotidedatabase sequences; BLASTX for nucleotide query sequences againstprotein database sequences; BLASTP for protein query sequences againstprotein database sequences; TBLASTN for protein query sequences againstnucleotide database sequences; and TBLASTX for nucleotide querysequences against nucleotide database sequences. The BLASTX program ispublicly available from NCBI and other sources (BLAST Manual, Altschul,S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J.Mol. Biol. 215:403-410 (1990)). The well-known Smith Waterman algorithmmay also be used to determine identity.

Preferred parameters for polypeptide sequence comparison include thefollowing: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453(1970); Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc.Natl. Acad. Sci. USA. 89:10915-10919 (1992); Gap Penalty: 12; and GapLength Penalty: 4. A program useful with these parameters is publiclyavailable as the “Ogap” program from Genetics Computer Group, located inMadison, Wis. The aforementioned parameters are the default parametersfor amino acid comparisons (along with no penalty for end gaps).

Preferred parameters for nucleic acid comparison include the following:Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970);Comparison matrix: matches=+10, mismatch=0; Gap Penalty: 50; Gap LengthPenalty: 3. Available as the Gap program from Genetics Computer Group,located in Madison, Wis. Given above are the default parameters fornucleic acid comparisons.

Ligand

As used herein, the term “ligand” refers to an agent that specificallybinds to a target RNA, preferably mRNA. As used herein the term“specific binding” means binding that is measurably different from anon-specific interaction. Specific binding can be measured, for example,by determining binding of an agent compared to binding of a controlagent, which generally is an agent of similar structure that does nothave binding activity, for example a RNA molecule of similar size thatlacks a specific binding sequence. Specific binding is present if theagent has measurably higher affinity for the target RNA than the controlagent. Specificity of binding can be determined, for example, bycompetition with a control agent that is known to bind to a target.E.g., specific binding of an agent can be demonstrated by competing forbinding with the same agent or a different agent specifically binding tothe target RNA. Specific binding can be indicated if the binding of amolecule is competitively inhibited by the second agent. The term“specific binding” as used herein, includes both low and high affinityspecific binding. Specific binding can be exhibited, e.g. by a lowaffinity binding agent having a Kd of at least about 1 mM and higher.Specific binding can also be exhibited by a high affinity binding agenthaving a Kd of at least about 1 μM and lower: The difference between ahigh and low affinity binding agent is about a factor 1,000. The agentmay bind the target RNA when the target RNA is in a native oralternative conformation, or when it is partially or totally unfolded ordenatured. According to the present invention, a ligand can be an agentthat binds anywhere on the target RNA, but preferably the ligand bindson the indicated nucleotide sequences of the target RNA. Ligands can bevirtually any agent, including without limitation metals, peptides,proteins, lipids, polysaccharides, small organic molecules, nucleotides(including non-naturally occurring ones) and combinations thereof

Messenger RNA

“Messenger RNA (mRNA)” as used herein refers to a temporarycomplementary copy of RNA of the antisense strand (anticoding strand ortemplate) of protein coding DNA. In eukaryotes it is usually transcribedas a relatively long pre-mRNA (also called primary transcript or hnRNA)which is then processed, still within the nucleus, to remove introns.Further post-transcriptional modifications can also occur. The maturemRNA is then transported into the cytoplasm where it is translated intoprotein on the ribosome. Furthermore, an mRNA generally comprises aregion that specifies the protein sequence, flanked on either side byuntranslated regions called 5′ and 3′ untranslated regions (5′UTR and3′UTR).

Antisense Nucleic Acid

“Antisense nucleic acid” as used herein refers to a RNA, DNA or PNAmolecule that is complementary to all or part of a target primarytranscript or mRNA and that blocks the translation of a targetnucleotide sequence.

DETAILED DESCRIPTION OF THE INVENTION

As a first aspect, the invention relates to a method for expressing aprotein or polypeptide of interest in a plant comprising the steps of:

a) providing a nucleic acid construct comprising a first nucleotidesequence that has at least 34% nucleotide sequence identity with thenucleotide sequence of SEQ ID No. 1, operably linked to a secondnucleotide sequence encoding a protein or polypeptide of interest andfurther operably linked to a heterologous promotor,

b) contacting a plant with said nucleic acid construct to obtain atransformed plant, and

c) subjecting said transformed plant to conditions leading to expressionof the protein or polyeptide of interest, and optionally recovering saidprotein or polypeptide.

According to the invention the nucleic acid construct comprises a firstnucleotide sequence that has at least 34% nucleotide sequence identityto the nucleotide sequence of SEQ ID No. 1 (using the BLAST algorithm ofBLASTN 2.2.1; 13-04-2001; gap penalties: existence 5, extension 2). Thenucleic acid construct according to the invention preferably comprises afirst nucleotide sequence that has at least 36%, more preferably atleast 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%nucleotide sequence identity to the nucleotide sequence of SEQ ID No. 1.In a particularly preferred embodiment of the invention the nucleic acidconstruct comprises a first nucleotide sequence that has 100% nucleotidesequence identity to the nucleotide sequence of SEQ ID No. 1. Thenucleotide sequence presented herein as SEQ ID No. 1 is the 5′ UTRsequence of the ntp303 gene of Nicotiana tabacum.

In a further embodiment the invention relates to a method for expressinga protein of interest in a plant comprising the steps of:

a) providing a nucleic acid construct comprising a first nucleotidesequence comprising a nucleotide sequence that has at least 46%nucleotide sequence identity to nucleotides 104-151 of the nucleotidesequence of SEQ ID No. 1 or a nucleotide sequence that has at least 51%nucleotide sequence identity to nucleotides 4-76 of the nucleotidesequence of SEQ ID No. 1 or a combination thereof, operably linked to asecond nucleotide sequence encoding a protein or polypeptide of interestand further operably linked to a heterologous promotor,

b) contacting a plant with said nucleic acid construct to obtain atransformed plant, and

c) subjecting said transformed plant to conditions leading to expressionof the protein or polypeptide of interest, and optionally recoveringsaid protein or polypeptide.

Preferably, said first nucleotide sequence comprises a nucleotidesequence that has at least 48%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 98% and particularly 100% nucleotide sequence identity tonucleotides 104-151 of the nucleotide sequence of SEQ ID No. 1 or anucleotide sequence that has at least 55%, more preferably at least 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% and particularly 100% nucleotidesequence identity to nucleotides 4-76 of the nucleotide sequence of SEQID No. 1 or a combination thereof.

In another embodiment of the above mentioned method the first nucleotidesequence comprises a nucleotide sequence that has at least 46%, 48%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% and particularly100% nucleotide sequence identity to nucleotides 104-151 of thenucleotide sequence of SEQ ID No. 1 or a nucleotide sequence that has100% nucleotide sequence identity to nucleotides 27-50 of the nucleotidesequence of SEQ ID No. 1 or a combination thereof. The nucleotides 27-50of the nucleotide sequence of SEQ ID No. 1 consist of a repeat of 8GAA-units. Preferably, said first nucleotide sequence comprises anucleotide sequence that consists of 7, 6, 5, 4 or 3 GAA units.

A nucleotide sequence according to the invention can be present in theform of RNA or in the form of DNA including genomic DNA, i.e. DNAincluding the introns, cDNA or synthetic DNA. The DNA may bedouble-stranded or single-stranded and if single-stranded may be thecoding strand or non-coding (anti-sense) strand. DNA or RNA with abackbone modified for stability or for other reasons are a further partof the invention. Moreover, DNA or RNA comprising unusual bases, such asinosine, or modified bases, such as tritylated bases are also a part ofthe invention. The nucleotide sequence may also be a allelic variant ofthe nucleotide sequence according to the invention. If desired, thenucleotide sequence can be prepared or altered synthetically so theknown codon preferences of the intended expression host canadvantageously be used. It has been shown for instance that the codonpreferences and GC content preferences of monocotyledons anddicotyledons differ (Murray et al., Nucl. Acids Res. 17: 477498 (1989)).

In a preferred embodiment of the method according to the invention thenucleic acid construct comprises a second nucleotide sequence encoding aprotein or polypeptide of interest that is operably linked to any one ofthe first nucleotide sequences as defined above. The protein orpolypeptide of interest can be a homologous protein or polypeptide, butin a preferred embodiment of the invention the protein or polypeptide ofinterest is a heterologous protein. A second nucleotide sequenceencoding a heterologous protein or polypeptide may be derived in wholeor in part from any source known to the art, including a bacterial orviral genome or episome, eukaryotic nuclear or plasmid DNA, cDNA orchemically synthesised DNA. The second nucleotide sequence mayconstitute an uninterrupted coding region or it may include one or moreintrons bounded by appropriate splice junctions, it can further becomposed of segments derived from different sources, naturally occurringor synthetic. The second nucleotide sequence encoding the protein orpolypeptide of interest according to the method of the invention ispreferably a full-length nucleotide sequence, but can also be afunctionally active part or other part of said full-length nucleotidesequence. The protein or polypeptide of interest may be a protein orpolypeptide conferring, for instance, insect resistance, droughtresistance, disease resistance, herbicide resistance, immunity, animproved intake of nutrients, minerals or water from the soil, or amodified metabolism in the plant. In another embodiment the plant isused for overproduction of the protein or polypeptide of interest. Thesecond nucleotide sequence encoding the protein or polypeptide ofinterest may also comprise signal sequences directing the protein orpolypeptide of interest when expressed to a specific location in thecell or tissue. Such signal sequences include, but are not limited to,sequences directing the protein or polypeptide of interest toorganelles, other plant cells or intercellular space. Furthermore, thesecond nucleotide sequence encoding the protein or polypeptide ofinterest can also comprise sequences which facilitate proteinpurification and protein detection by for instance Western blotting andELISA (e.g. c-myc or polyhistidine sequences).

The protein or polypeptide of interest may have industrial or medicinal(pharmaceutical) applications. Examples of proteins or polypeptides withindustrial applications include enzymes such as e.g. lipases (e.g usedin the detergent industry), proteases (used inter alia in the detergentindustry, in brewing and the like), cell wall degrading enzymes (suchas, cellulases, pectinases, β-1,3/4- and β-1,6-glucanases,rhamnoga-lacturonases, mannanases, xylanases, pullulanases,galactanases, esterases and the like, used in fruit processing winemaking and the like or in feed), phytases, phospholipases, glycosidases(such as amylases, (β-glucosidases, arabinofuranosidases, rhamnosidases,apiosidases and the like), dairy enzymes (e.g. chymosin). Mammalian, andpreferably human, proteins or polypeptides and/or enzymes withtherapeutic, cosmetic or diagnostic applications include, but are notlimited to, insulin, serum albumin (HSA), lactoferrin, hemoglobin α andβ, tissue plasminogen activator (tPA), erythropoietin (EPO), tumornecrosis factors (TNF), BMP (Bone Morphogenic Protein), growth factors(G-CSF, GM-CSF, M-CSF, PDGF, EGF, and the like), peptide hormones (e.g.calcitonin, somatomedin, somatotropin, growth hormones, folliclestimulating hormone (FSH) interleukins (IL-x), interferons (IFN-y). Alsoincluded are bacterial and viral antigens, e.g. for use as vaccines,including e.g. heat-labile toxin B-subunit, cholera toxin B-subunit,envelope surface protein Hepatitis B virus, capsid protein Norwalkvirus, glycoprotein B Human cytomegalovirus, glycoprotein S, interferon,and transmissible gastroenteritis corona virusreceptors and the like.Further included are genes coding for mutants or analogues of the saidproteins.

In an embodiment of the invention the nucleic acid construct furthercomprises a promotor for control and initiation of transcription of thesecond nucleotide sequence. The promoter preferably is capable ofcausing expression of the second nucleotide sequence in the host cell ofchoice. Said promoter, e.g. pollen-specific or heterologous, is operablylinked to any one of the nucleotide sequences mentioned above. In apreferred embodiment of the invention the promoter is a plant promotor,i.e. a promoter capable of initiating transcription in plant cells.Plant promotors as used herein include tissue-specific,tissue-preferred, cell-type-specific, inducible and constitutivepromotors. Tissue-specific promotors are promoters which initiatetranscription only in certain tissues and refer to a sequence of DNAthat provides recognition signals for RNA polymerase and/or otherfactors required for transcription to begin, and/or for controllingexpression of the coding sequence precisely within certain tissues orwithin certain cells of that tissue. Expression in a tissue specificmanner may be only in individual tissues or in combinations of tissues.In a preferred embodiment of the invention the expression is pollen orseed specific, i. e. the expression is specific to pollen or seeds only.Pollen-specific and seed-specific promoters include, but are not limitedto, promotors of the pollen-specific genes ntp303 (N. tabacum) and zm13(Z. mays) and the seed-specific genes dc8 (D. carota), rab17 (Z. mays),rab16b (O. sativa) and em (T. aestivum). The group of tissue-specificpromoters are reviewed by Edwards, J. W. & Cornzzi, G. M., Annu Rev.Genet. 24, 275-303 (1990) and include, but are not limited to,embryo-specific promotors such as the promoters of the embryonic storageproteins soybean .beta.-conglycinin gene, legumin genes from commonbean, .beta.phaseolin gene and napin and cruciferin genes from rapeseed,endosperm-specific promotors such as the promoters of maize zein genes,wheat glutenin genes and barley hordein genes, fruit-specific promotorssuch as the promotor of the tomato ethylene-responsive E8 gene,tuber-specific promotors such as the class-I patatin promotor of potatoand leaf-specific promotors such as the promotors ofribulose-1,5-biphosphate carboxylase small subunit gene and thechlorophyll a/b binding protein gene.

Tissue-preferred promotors are promoters that preferentially initiatetranscription in certain tissues, such as leaves, roots, stems, flowersor seeds.

Cell-type-specific promoters are promoters that primarily driveexpression in certain cell types in one or more organs, for example,vascular cells in roots or leaves. Inducible promoters are promotersthat are capable of activating transcription of one or more DNAsequences or genes in response to an inducer. The DNA sequences or geneswill not be transcribed when the inducer is absent. Inducers known inthe art include high salt concentrations, cold, heat or toxic elementsand include pathogens or disease agents such as virusses. Or inducerscan be chemical agents such as herbicides, proteins, growth regulators,metabolites or phenolic compounds. The inducer can also be anillumination agent such as darkness and light at various modalitiesincluding wavelength, intensity, fluence, direction and duration.Activation of an inducible promoter is established by application of theinducer. The group of generally inducible promotors includes, but is notlimited to, the hsp70 heat shock promoter of Drosphilia melanogaster, acold inducible promoter from Brassica napus and an alcohol dehydrogenasepromoter which is induced by ethanol. Specific plant inducible promotorsinclude, but are not limited to, the tetracycline-inducible promotor andthe .alpha.-amylase promotor.

Constitutive promoters are promoters that are active under manyenvironmental conditions and in many different tissue types. The groupof constitutive promotors includes, but is not limited to, the 35Spromotor or 19S promotor of the cauliflower mosaic virus (CaMV), theubiquitin promotor, the coat promoter of TMV, the cassava vein mosaicvirus promotors (CsVMV), the rice actin-I promotor and regulatoryregions associated with Agrobacterium genes, such as nopaline synthase(Nos), mannopine synthase (Mas) or octopine synthase (Ocs).

The nucleic acid construct according to the invention is preferably avector, in particular a plasmid, cosmid or phage or nucleotide sequence,linear or circular, of a single or double stranded DNA or RNA, derivedfrom any source, in which a number of nucleotide sequences have beenjoined or recombined into a unique construction which is capable ofintroducing any one of the nucleotide sequences of the invention insense or antisense orientation into a cell, in particular a plant cell.The choice of vector is dependent on the recombinant procedures followedand the host cell used. The vector may be an autonomously replicatingvector or may replicate together with the chromosome into which it hasbeen integrated. Preferably, the vector contains a selection marker.Useful markers are dependent on the host cell of choice and are wellknown to persons skilled in the art. In case the protein is to beobtained from leaves or roots, infection of cells with a viral vectorhas the advantage that a large proportion of the targeted cells canreceive the nucleic acid. Additionally, molecules encoded within theviral vector, e.g., by a cDNA contained in the viral vector, areexpressed efficiently in cells which have taken up viral vector nucleicacid. Suitable vectors which can be delivered using the presently knownprocedures include, but are not limited to, herpes simplex virusvectors, adenovirus vectors, papovavirus vectors (such as humanpapillomavirus vectors, polyomavirus vectors, SV40 vectors),adeno-associated virus vectors, retroviral vectors, pseudorabies virus,alpha-herpes virus vectors, and the like. A thorough review of viralvectors, particularly viral vectors suitable for modifyingnonreplicating cells, and how to use such vectors in conjunction withthe expression of polynucleotides of interest can be found in the bookVIRAL VECTORS: GENE THERAPY AND NEUROSCIENCE APPLICATIONS (Ed. Caplittand Loewy, 1995). Agrobacterium-based plasmid vectors are preferred forstable transformation of nucleic acid constructs in a plant genome. Thechoice of the transformation vector is dependent on the followedtransformation procedure and the used host cell. Binary Ti vectors whichcan be used for Agrobacterium-mediated gene transfer include pBIN19,pC22, pGA482 and pPCV001.

A recombinant host cell, such as a mammalian (with the exception ofhuman), plant, animal, insect, fungal or bacterial cell, containing oneor more copies of a nucleic acid construct according to the invention isan additional subject of the invention. By host cell is meant a cellwhich contains a nucleic acid construct such as a vector and supportsthe replication and/or expression of the nucleic acid construct.Examples of suitable bacteria are Gram positive bacteria such as severalspecies of the genera Bacillus, Streptomyces and Staphylococcus or Gramnegative bacteria such as several species of the genera Escherichia andPseudomonas. In the group of fungal cells preferably yeast cells areused. Expression in yeast can be achieved by using yeast strains such asPichia pastoris, Saccharomyces cerevisiae and Hansenula polymorpha.Furthermore, insect cells such as cells from Drosophila and Sf9 can beused as host cells. Alternatively, a suitable expression system can be abaculovius system or expression systems using mammalian cells such asCHO, COS or Bowes melanoma cells. For transformation procedures inplants, suitable bacteria include Agrobacterium tumefaciens andAgrobacterium rhizogenes.

Another aspect of the invention relates to a plant that is geneticallymodified, preferably by the method of the invention, in that the plantcomprises a nucleic acid construct as herein defined above. The nucleicacid construct preferably is a construct containing nucleic acidsequences that are manipulated or modified in vitro or at least explauta. As such, the nucleic acid construct preferably provides theplant with a combination of nucleic acid sequences which is not found innature. The nucleic acid construct preferably is stably maintained,either as a autonomously replicating element, or, more preferably, thenucleic acid construct is integrated into the plant's genome, in whichcase the construct is usually integrated at random positions in theplant's genome, for instance by nonhomologuous recombination. Plantsthat are preferred in the invention include tobacco, potato, sugar,beet, soja, maize, rice, lupin, alfalfa, Arabidopsis and Brassica.Stably transformed (transgenic) plants or plant cells are produced byknown methods. The term stable transformation refers to exposing plants,tissues or cells thereof to methods to transfer and incorporate foreignDNA into the plant genome. These methods include, but are not limitedto, Agrobacterium tumefaciens-mediated gene transfer, transfer ofpurified DNA via microparticle bombardment, electroporation ofprotoplasts and microinjection or use of silicon fibers to facilitatepenetration and transfer of DNA into the plant cell. Dicotyledonousplants are most frequently transformed by Agrobacterium-mediated genetransfer such as for instance by co-culture of regenerating plantprotoplasts or cell cultures with Agrobacterium tumefaciens. In general,when Agrobacterium tumefaciens is used for transformation, thetransformation vectors are preferably cointigrating vectors or binaryvectors. Dicotyledonous plants can furthermore be transformed bytransformation of leaf discs, by protoplast transformation bypolyethylene glycol-induced DNA transfer, electroporation, sonication ormicroinjection as well as transformation of intact cells or tissues bymicro- or macroinjection into tissues or embryos, tissueelectroporation, incubation of dry embryos in DNA-containing solution,vacuum infiltration of seed and biolistic gene transfer.Monocotyledonous plants are transformed via for example particlebombardment, electrically or chemically induced DNA incorporation intoprotoplasts, electroporation of partially permeabilized cells,macroinjection of DNA into inflorescences, microinjection of DNA intomicrospores and pro-embryos, the introduction of DNA into germinatingpollen and DNA integration into embryos by swelling.

An alternative method to express a protein or polypeptide of interest inplants relies on transient expression from virus-based vectors. It isknown that viruses replicate with high efficiency and, in some cases,can infect the entire host plant, creating the potential to express aprotein or polypeptide of alternative method are tobamovirus, potexvirusand potyvirus.

Alternatively, next to the expression in host cells such as plant cellsthe protein or polypeptide of interest can be produced in cell-freetranslation systems using RNAs derived from the nucleic acid constructsof the present invention.

In the method according to the invention the nucleic acid construct canfurther optionally comprise interest in large amounts. Vectors that canbe used in this other regulatory elements known in the art that aresuitable in said method. These include, but are not limited to, elementspresent in the 5′ UTR, 3′ UTR and coding nucleotide sequences ofhomologous and/or heterologous nucleotide sequences, including the IronResponsive Element (IRE), Translational cis-Regulatory Element (TLRE) oruORFs in 5′ UTRs and poly(U) stretches in 3′ UTRs. Preferably, saidregulatory elements are operably linked to the nucleotide sequences andpromotors according to the invention.

When a transformed tissue or cell (e.g., pieces of leaf, stem segments,roots, but also protoplasts or plant cells cultivated by suspension) isobtained with the method according to the invention, whole plants can beregenerated from said transformed tissue or cell in a suitable medium,which optionally may contain antibiotics or biocides known in the artfor the selection of transformed cells.

Resulting transformed plants are preferably identified by means ofselection. The nucleic acid construct according to the inventiontherefore preferably also comprises a marker gene which can provideselection or screening capability in a treated plant. Selectable markersare generally preferred for plant transformation events, but are notavailable for all plant species. Suitable selectable markers can beantibiotic or herbicide resistant genes which, when inserted in somecells of a plant in culture, would confer on those cells the ability towithstand exposure to an antibiotic or a herbicide. Another type ofmarker gene is one that can be screened by histochemical or biochemicalassay, even though the gene cannot be selected for. A suitable markergene found useful in such plant transformation experience is the GUSgene. Jefferson et al., EMBO J., 6: 3901-3907 (1987), disclose thegeneral protocol for a GUS assay. The GUS gene encodes an enzyme thatcatalyzes the cleavage of 5-bromo-4-chloro-3-indolyl glucuronide, asubstrate that has a blue color upon cleavage. Thus, the use of a GUSgene provides a convenient assay for the detection of the expression ofintroduced DNA in plants by histochemical analysis of the plants. In anexample of a transformation process, the gene sought to be expressed inthe plant could be coupled in tandem with the GUS gene. The tandemconstruct could be transformed into plants, and the resulting plantscould be analyzed for expression of the GUS enzyme. Another example of amarker gene is luciferase. An advantage of this marker is thenon-destructive procedure of application of the substrate and thesubsequent detection. The transformed plants can also be identified byexpression of the gene of interest.

In a next step the transformed plant, part or cell thereof is subjectedto conditions leading to expression of the protein or polypeptide ofinterest, and optionally recovering said protein or polypeptide.Recovering steps depend on the expressed protein or polypeptide and thehost cell used but can comprise isolation of the protein or polypeptide.When applied to a protein/polypeptide, the term “isolation” indicatesthat the protein is found in a condition other than its nativeenvironment. In a preferred form, the isolated protein is substantiallyfree of other proteins, particularly other homologous proteins. It ispreferred to provide the protein in a greater than 40% pure form, morepreferably greater than 60% pure form. Even more preferably it ispreferred to provide the protein in a highly purified form, i.e.,greater than 80% pure, more preferably greater than 95% pure, and evenmore preferably greater than 99% pure, as determined by SDS-PAGE. Ifdesired, the second nucleotide sequence may be ligated to a heterologousnucleotide sequence to encode a fusion protein to facilitate proteinpurification and protein detection on for instance Western blot and inan ELISA.

Suitable heterologous sequences include, but are not limited to, thenucleotide sequences encoding for proteins such as for instanceglutathione-S-transferase, maltose binding protein, metal-bindingpolyhistidine, green fluorescent protein, luciferase andbeta-galactosidase. The protein may also be coupled to non-peptidecarriers, tags or labels that facilitate tracing of the protein, both invivo and in vitro, and allow for the identification and quantificationof binding of the protein to substrates. Such labels, tags or carriersare well-known in the art and include, but are not limited to, biotin,radioactive labels and fluorescent labels.

In a particularly preferred embodiment of the method according to theinvention the plant that is used for the expression of the protein orpolypeptide of interest is a doubled haploid (homozygous) transgenicNicotiana tabacum plant silenced for Ntp303. The transgenic plantoriginates from Nicotiana tabacum cv. Petit havana which was transformedwith antisense ntp303 and selected for via anther culture. Fortyindependent transformed plantlets were generated from anthers by antherculture in vitro. Four of them were doubled haploids and could producehaploid pollen. The morphological and agronomic characters of thesedoubled haploids with 1 n gametes were compared with those of their wildtype parent. The transgenic plant and the flowers it produces areexactly the same as the wild type. Moreover, it gives a normal amount ofpollen, but crossings give no offspring (i.e. the plant is malesterile). However, the pollen can be germinated in vitro and its pollentube growth is similar to that of wild type pollen. In plantagermination of the pollen is poor and the few pollen tubes that areformed stop growing after 10 mm of growth into the style.

Preferably, the protein or polypeptide of interest is expressed inpollen tubes or seed of a plant used in the method according to theinvention. Mass culture of pollen tubes in vitro can be established in agermination chamber according to the specifications of Schrauwen andLinskens (1967) Acta Bot. Neerl. 16 (5), 177-179.

Furthermore, a part of the invention is a doubled haploid homozygoustransgenic Nicotiana tabacum plant silenced for Ntp303. Preferably, saidtransgenic plant comprises any one of the nucleic acid constructdescribed above. Propagation, harvest and tissue material of saidtransgenic plant, including, but not limited to, leafs, roots, shootsand flowers, are also a part of the invention.

Yet a further aspect of the invention pertains to ligands thatspecifically bind to a first nucleotide sequence according to theinvention comprised in a RNA molecule, thereby regulating thetranslation of a second nucleotide sequence encoding a protein orpolypeptide of interest operably linked to the first nucleotidesequence.

In one embodiment of the invention the ligand of the inventionspecifically binds to a first nucleotide sequence comprised in a RNAmolecule, whereby the first nucleotide sequence has at least 34%nucleotide sequence identity to the nucleotide sequence of SEQ ID No. 1.The nucleotide sequence represented by SEQ ID No. 1 is in the DNA form,but the skilled worker understands that when this nucleotide sequence isin the RNA form thymine (T) has to be replaced by uracil (U).Preferably, the ligand of the invention specifically binds to a firstnucleotide sequence comprised in a RNA molecule, whereby the firstnucleotide sequence has at least 36%, more preferably at least 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% nucleotidesequence identity to the nucleotide sequence of SEQ ID No. 1. In aparticularly preferred embodiment of the invention the ligand of theinvention specifically binds to a first nucleotide sequence comprised ina RNA molecule, whereby the first nucleotide sequence has 100%nucleotide sequence identity to the nucleotide sequence of SEQ ID No. 1.The nucleotide sequence presented herein as SEQ ID No. 1 is as mentionedabove the 5′ UTR sequence of the ntp303 gene of Nicotiana tabacum.

In a further embodiment of the invention the ligand specifically bindsto a first nucleotide sequence comprised in a RNA molecule, whereby thefirst nucleotide sequence comprises a nucleotide sequence that has atleast 46% nucleotide sequence identity to nucleotides 104-151 of thenucleotide sequence of SEQ ID No. 1. Preferably, the ligand specificallybinds to a first nucleotide sequence comprised in a RNA molecule,whereby the first nucleotide sequence comprises a nucleotide sequencethat has at least 48%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,98% and particularly 100% nucleotide sequence identity to nucleotides104-151 of the nucleotide sequence of SEQ ID No. 1. In a preferredembodiment of the invention the ligand specifically binds to a firstnucleotide sequence (or a part thereof) comprised in a RNA molecule,whereby the first nucleotide sequence is transcribed from the nucleotidesequence that has at least 46% nucleotide sequence identity tonucleotides 104-151 of the nucleotide sequence of SEQ ID No. 1.

In another embodiment of the invention the ligand specifically binds toa first nucleotide sequence comprised in a RNA molecule, whereby thefirst nucleotide sequence comprises a nucleotide sequence that has atleast 51% nucleotide sequence identity to nucleotides 4-76 of thenucleotide sequence of SEQ ID No. 1. Preferably, the ligand specificallybinds to a first nucleotide sequence comprised in a RNA molecule,whereby the first nucleotide sequence comprises a nucleotide sequencethat has at least 55%, more preferably at least 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 98% and particularly 100% nucleotide sequence identity tonucleotides 4-76 of the nucleotide sequence of SEQ ID No. 1. In apreferred embodiment of the invention the ligand specifically binds to afirst nucleotide sequence (or a part thereof) comprised in a RNAmolecule, whereby the first nucleotide sequence is transcribed from thenucleotide sequence that has at least 51% nucleotide sequence identityto nucleotides 4-76 of the nucleotide sequence of SEQ ID No. 1.

In yet another embodiment of the invention the ligand according to theinvention specifically binds to a first nucleotide sequence comprised ina RNA molecule, whereby the first nucleotide sequence comprises anucleotide sequence having the nucleotide sequence of nucleotides 27-50of the nucleotide sequence of SEQ ID No. 1.

In a preferred embodiment of the invention the ligand specifically bindsto a first nucleotide sequence (or a part thereof) comprised in a RNAmolecule, whereby the first nucleotide sequence is transcribed from thenucleotide sequence having the nucleotide sequence of nucleotides 27-50of the nucleotide sequence of SEQ ID No. 1.

In a preferred embodiment of the invention the ligand specifically bindsto a first nucleotide sequence (or a part thereof) comprised in an atleast partly double stranded RNA molecule, whereby the first nucleotidesequence is transcribed from the nucleotide sequence having thenucleotide sequence of nucleotides 14-21 and the complementary strandhaving the nucleotide sequence of nucleotides 59-66 of the nucleotidesequence of SEQ ID No. 1.

If desired, a combination of the ligands mentioned above can be used.

Ligands according to the invention can be metals, peptides, proteins,lipids, polysaccharides, small organic molecules, nucleotides (includingnon-naturally occurring ones) and combinations thereof. Preferably, theligands according to the invention are peptides, proteins, nucleotidesor combinations thereof.

Metals that can be used as ligands according to the invention include,but are not limited to, Mg²⁺, K⁺ (G-quartet), Fe²⁺, Cu²⁺ and PtCl₂.

Lipids that can be used as ligands according to the invention include,but are not limited to, lipochitooligosaccharides (LCOs), cationiclipids, anionic lipids and zitterionic lipids.

Polysaccharides that can be used as ligands according to the inventioninclude, but are not limited to, lipochitooligosaccharides (LCDs).

Small organic molecules that can be used as ligands according to theinvention include, but are not limited to, caffeine, pophyrine,peptides, haem, aromatic structures and OH (hydroxyl) in combinationwith NH₂ groupes in f.e. neomycine. Preferably, these small organicmolecules have a molecular weight of more than 50 yet less than about2,500 daltons, and most preferably less than about 400 daltons.

Nucleotides that can be used as ligands according to the inventioninclude but are not limited to antisense nucleic acid molecules. Theantisense nucleic acid molecules can be selected from antisense RNAmolecules, antisense DNA molecules and derivatives thereof, e.g. HNA,fosforothioate-DNA, antisense PNA molecules with lengths of 8-20 units.

Peptides with lengths up to 60 AA (amino acids) that can be used asligands according to the invention.

An important group of ligands that can be used according to theinvention encompasses proteins that are able to interact with RNA. TheseRNA binding proteins (RBP) appear to mediate inter alia the processingof pre-mRNAs, the transport of mRNA from the nucleus to the cytoplasm,mRNA stabilization, the translational efficiency of mRNA, and thesequestration of some mRNAs. RNA binding proteins that can be used asligands according to the invention include but are not limited toproteins comprising a RNA binding protein motifs such as inter alia theheterogenous nuclear) ribonucleoprotein (RNP) motif, Arg-rich motif, RGGbox, KH motif and double-stranded RNA-binding motif (for review see Burdand Dreyfuss, Science 265:615-621 (1994). These motifs recognize bothsequence and structure dependent RNA elements. Furthermore, proteinssuch as inter alia RNA hairpin-binding factors, proteins with a RNArecognition motif, GAA-binding proteins, RNA-binding proteins with aserine-rich domain, heat shock proteins and cellular nucleic acidbinding proteins can be used as ligands.

Preferably, the ligand according to the invention is naturally occurringin the host cell, wherein the first nucleotide sequence comprised in aRNA molecule is present. Alternatively, the ligand can also beintroduced into the host cell by methods known in the art.

Use of a ligand according to the invention for regulating translation ofa second nucleotide sequence encoding a protein or polypeptide ofinterest, the second nucleotide sequence operably linked to the firstnucleotide sequence is also a part of the present invention. The secondnucleotide sequence can encode a homologous or heterologous protein orpolypeptide according to the invention. The specific binding of theligand to the first nucleotide comprised in a RNA molecule can on theone hand lead to a decrease of translation of the second nucleotidesequence encoding the protein or polypeptide of interest. Alternatively,the binding can also lead to an increase of translation. An increase intranslation is mediated by the binding of inter alia a protein orpolypeptide that specifically binds to a double stranded piece of RNA inthe H-I structure (see page 32 lines 2-4). This protein contains anamino acid sequence Ser (Arg/Lys) (Arg/Lys) Xaa (Ala/Pro) Arg Lys(Asn/Gln/His) Lys (SEQ ID NO: 14) in which Xaa is any amino acid and theamino acids in parentheses represent one of two or three alternativeamino acids that are considered to be conservative amino acidsubstitutions.

EXAMPLES

Materials and Methods

Plant Material

Greenhouse-grown plants of Nicotiana tabacum L. cv. Petit Havana SRIwere used as the source of pollen and leaf tissue for microprojectilebombardment. To assess the transient expression of the differentchimeric genes during pollen development, immature pollen at thelate-bicellular stage were aseptically isolated from flower buds of 35mm length in M1 medium as previously described (Tup et al., (1991) Sex.Plant Reprod. 4(4): 284-287). Transient expression of different genefusion constructs during pollen tube growth was measured using maturepollen which were isolated from dehiscent tobacco flowers (Herpen etal., (1992) Sex. Plant Reprod. 5, 304-309).

After isolation, the pollen pellet was suspended in 100 μl M1-medium ata density of 10⁸ cells/ml. To fixate the pollen for particlebombardment, the pollen suspension was pipetted onto the surface of asterile Hybond-N membrane (Amersham) that was placed on 1% agarsolidified M1 medium. Following bombardment, the membrane containinglate-bicellular or mature pollen was soaked in 10 ml of M1 medium orRead-medium (Read et al., (1993) Protoplasma 174, 101-115.),respectively. The late-bicellular pollen was incubated at 25° C. in thedark at vigorous shaking. After centrifugation, the mature pollen wassuspended in a 10-ml tube containing 0.5 ml Read medium followed by a 20hours incubation in the dark. Treatment of leaf tissue before and afterbombardment was performed as described by Hamilton et al., (1992) PlantMol. Biol. 18, 211-218.

In all cases, bombardments were done within 60 min of placing plantmaterials onto the solidified medium.

Preparation of Gene Fusion Constructs Containing Different UTRs

In all constructs, either a modified version of the firefly luciferasecoding region, luc⁺, or luciferase cDNA from Renilla reniformis (rluc)was used as the reporter gene. The luc⁺ cDNA was amplified by thepolymerase chain reaction (PCR) on the pGL3 vector (Promega) using aforward sequence-specific primer which introduced a NcoI site at the 5′end (5′-ATATCCATGGAAGACGCC; NcoI site underlined; SEQ ID No. 2) and areverse sequence-specific primer which introduced a BamHI site at the 3′end (5′-ATATGGATCCTTACACGGCGATC; BamHI site underlined; SEQ ID No. 3).The rluc cDNA was amplified by PCR on the pRL-SV 40 vector (Promega)using the following sequence-specific primers:5′-GTGTCCATGGATGACTTCGAAAG (NcoI site underlined; SEQ ID No. 4) and5′-GTGTGGATCCTTATTGTTCATTTTTGAG (BamHI site underlined; SEQ ID No.5).For construction of ^(35S)syn44 5′/35S 3′, the PCR product of luc⁺ wasdigested with NcoI and BamHI and, after removal of the luciferase gene,ligated into the NcoI and BamHI sites in pRTS2LUC (Bate et al., (1996)Plant J., 10(4), 613-623). pRTS2LUC (kindly provided by Dr. David Twell)contained the CaMV35S promoter (Topfer et al., 1987), a 44 basepairslong synthetic polylinker (designated as syn44 5′ in this article), theluciferase cDNA (Ow et al., 1986) and the CaMV 35S 3′ untranslatedregion (Topfer et al., (1987) Nucl. Acids. Res. 15, 5890).

An almost identical construct was built, ^(35S R)syn44 5′/35S 3′, inwhich the luc⁺ gene was replaced by the rluc coding region. To obtain agene fusion construct containing both ntp303 UTRs and the CaMV35Spromoter (pRH1-1 ^(35S)303 5′/303 3′), the syn44 5′ UTR was removed frompRH4-1 using XhoI and NcoI restriction enzymes. The ntp303 5′ UTR wasamplified by PCR on the ntp303 genomic clone (Weterings et al., (1995)Sex. Plant Reprod. 8(1) 11-17) using the following primers withrestriction sites incorporated into the 5′ end:5′-GTGTCTCGAGCAAGCTCTAGCAGGAAG (XhoI site underlined; SEQ ID No.6) and5′-GTGTCCATGGGACGTTGTTTTTTTATTC (NcoI site underlined; SEQ ID No. 7).Following the PCR, the ntp303 5′ UTR was obtained with XhoI-NcoIdigestion and ligated in the ^(35S)syn44 5′/35S 3′ construct lacking thesyn44 5′ UTR to create the plasmid ^(35S)303 5′/35S 3′. Theoligonucleotides 5′-ATATGGATCCATTCTGTAATGATCAATCTG (BamHI siteunderlined; SEQ ID No. 8) and 5′-ATATGAGCTCATTTAATGTTTTGTCCTA (SacI siteunderlined; SEQ ID No. 9) were used to generate the ntp303 3′ UTR usingthe ntp303 genomic clone as template. This PCR fragment was digestedwith BamHI and Sad and cloned into ^(35S)303 5′/35S 3′ to replace theCaMV 35S 3′ UTR and to create ^(35S)303 5′/303 3′.

Gene fusion constructs containing the ntp303 promoter were made asfollow. Using the genomic clone of ntp303 as template, a 578 basepairslong promoter fragment, including the transcription initiation site, wasamplified (Weterings et al., 1995) using the primers5′-ATATAAGCTTGATACACTCGCAACGTGTGT (HindIII site underlined; SEQ ID No.10) and 5′-ATATCTCGAGGAGCTTGCACTATTCACCAT (XhoI site underlined; SEQ IDNo. 11). The amplified ntp303 promoter fragment, that included theregion which has been demonstrated to reflects the minimal upstreamregion of the ntp303 gene that is capable to direct pollen expression,was digested with HindIII and XhoI and, after removal of the CaMV 35Spromoter, ligated into ^(35S R)syn44 5′/35S 3′, ^(35S)303 5′/35S 3′ and^(35S)303 5′/303 3′ to create ^(R)syn44 5′/35S 3′, 303 5′/35S 3′ and 3035′/303 3′, respectively. To obtain a construct containing the ntp303promoter, the ntp303 UTRs and the Renilla luciferase cDNA (^(R)3035′/303 3′), the luc⁺ gene was digested from 303 5′/303 3′ using NcoI andBamHI, after which the rluc coding region was ligated into the NcoI andBamHI sites. The constructs which were regulated by the ntp303 promoterand which expressed the luc⁺ gene contained a longer version of thesynthetic linker than which was used in all the other constructs. This99-basepairs long synthetic leader was obtained by PCR using thepNBL52-44 plasmid as the template (a kindly gift from Dr. David Twell).This fragment, designated as syn99 5′ in this article, was amplifiedusing the following primers: 5′-GTGTCTCGAGTTGCAATTGGATCC (XhoI siteunderlined; SEQ ID No. 12) and 5′-GTGTCCATGGCCGCGGG (NcoI siteunderlined; SEQ ID No. 13). After removal of the ntp303 5′ UTR from 3035′/35S 3′ and 303 5′/303 3′, the syn99 5′ UTR was cloned into the XhoIand NcoI sites creating the syn99 5′/35S 3′ and syn99 5′/303 3′constructs, respectively.

Constructs containing modifications of the ntp303 5′ UTR (A 5′ UTR) wereall obtained by PCR using the ntp303 5′ UTR in the 303 5′/35S 3′construct as starting-material. Fragments which were obtained by PCRwere sequenced completely to exclude mismatches within the sequences.All constructs used for transient expression were in the pUC19 plasmid.

Microprojectile Bombardment

Microcarriers, rupture disks and macrocarriers were obtained fromBio-Rad. Preparation and coating of the microcarriers was performedaccording the manufacture's manual (Bio-Rad). For biolistictransformation of late bicellular pollen and mature pollen, we used perbombardment 250 μg gold particles with a size of 1 μm and 1.6 μm,respectively. The microcarriers were coated with a total amount of 1 μgDNA containing 0.7 μg test plasmid DNA and 0.3 μg normalisation plasmidDNA. Test plasmids containing the ntp303 promoter and the luc⁺ gene, theCaMV 35S promoter and the luc⁺ gene, or the ntp303 promoter and the rlucgene were Co-precipitated with the normalisation plasmids ^(R)syn445′/35S 3′, ^(35S R)syn44 5′/35S 3′ and syn44 5′/35S 3′, respectively.syn44 5′/35S 3′ is identical to syn99 5′/35S 3′, with the exception ofcontaining the SYN44 5′ leader instead of the SYN99 5′ leader.Microprojectile bombardment was performed using the helium-drivenPDS-1000/He System of Bio-Rad. For biolistic transformation of pollenand leaves, the following bombardment parameters were used: a targetdistance of 6 cm, a gap distance of ¼ inch, a microprojectile/stoppingscreen distance of 8 mm, a chamber vacuum of 28 mm Hg, and a burstpressure of the rupture disks of 1100 psi.

Luciferase Assays

After particle bombardment and incubation of the tissues, quantitativedetermination of transient expression of the different gene fusionconstructs was performed using the commercial available Dual-Luciferase™Reporter Assay System (Promega). In this assay, the activities of theLUC+ and RLUC luciferases were measured sequentially from a singlesample extract using a luminometer provided with two auto-injectors(Wallac 1420 VICTOR²™, EG&G Wallac). Preparation of the buffers used inthe assay was performed according the manufacture's manual (Promega).After incubation, the developing pollen were transferred into a 10-mlGreiner tube and collected by centrifugation for 2 minutes at 2,500 rpm.Germinating pollen were collected by centrifugation for 5 minutes at1,000 rpm. In all cases, the pollen pellet was resuspended in 100 μl 1×passive lysis buffer (Promega) and grinded in liquid nitrogen. Thepollen extracts were stored at −70° C. until use for the luciferaseactivity assay. Extracts (10 μl) were pipetted in a microtiter plateafter which the plate was placed in the luminometer. 100 μl ofLuciferase Assay Reagent II (Promega) was automatically injectedfollowed, after 2 seconds, by a counting of the photons for 10 seconds.Immediately after the quantification of the LUC⁺ luminescence, thereaction was quenched, and the RLUC reaction was initiated afterautomatically addition of 100 μl of Stop&Glo™ Reagent (Promega). RLUCluminescence was also measured for 10 seconds, 2 seconds afterinjection. To compensate for variability of the expression of a sametest reporter gene between independent experiments, the ratio of LUC⁺:RLUC was determined. For each construct, at least six independentbombardment were performed.

EXAMPLE 1

UTR Gene Fusion Constructs

Several gene fusion constructs containing the ntp303 promoter, thefirefly luciferase reporter gene and different combinations of 5′- and3′ UTRs were built to investigate the ability of the UTRs of ntp303 tomodulate gene expression during pollen development and pollen tubegrowth (FIG. 1). The names of the constructs refer to their 5′ UTR and3′ UTRs (5′ UTR/3′ UTR). The abbreviation ‘35S’ or ‘R’ which is given inuppercase before a construct name indicates that the construct containsthe CaMV 35S promoter or Renilla luciferase coding region, respectively.

The UTR gene fusion constructs were introduced by particle bombardmentinto developing or mature pollen. Their transient expression wasmeasured after a period of 20 hours ill vitro development or germinationby luminescence measurements. To correct for differences in bombardmentefficiencies, a second construct was co-bombarded containing the ntp303promoter, a synthetic 5′ UTR (syn44 5′), the cauliflower mosaic virustermination sequence (35S 3′) and a luciferase reporter gene fromRenilla (^(R)syn44 5′/35S 3′). The transient expression value of thefirefly luciferase construct was normalized to the value of the Renillaluciferase construct.

The effect of ntp303 UTRs on transient expression during pollendevelopment and pollen tube growth was investigated by comparing theexpression level of a construct containing the ntp303 UTRs (303 5′/3033′) with that of constructs containing control UTRs (the syn99 5′ orsyn44 5′ UTR and the 35S 3′ UTR). After 20 hours of pollen tube growth,the expression of 303 5′/303 3′ was approximately 60- and 6-fold higherthan that of syn99 5′/35S 3′ and syn44 5′/35S 3′, respectively (FIG. 2b). The differences in the expression level were already observed intubes of pollen which were bombarded 5 hours before. Such largedifferences in expression level were not observed in developing pollenincubated for 20 hours after bombardment of the ntp303 UTRs construct.Here, the ntp303 UTRs gave rise to an expression level that wasapproximately 4-fold higher than that of syn99 5′/35S 3′ UTRs andslightly lower than that of syn44 5′/35S 3′ (FIG. 2 a). The expressionlevels of the constructs containing the control UTRs were more or lessthe same during pollen development and pollen tube growth (FIGS. 2 a and2 b). This clearly illustrates that expression mediated by these controlUTRs is independent of the developmental stage in which they weretested.

To examine whether the 5′ UTR or the 3′ UTR of the ntp303 mRNAdetermines the level of gene expression during pollen development andpollen tube growth, transient expression levels of gene fusionconstructs containing the ntp303 5′/35S 3′ or the syn44 5′/ntp303 3′UTRs were compared with that of syn44 5′/35S 3′. During pollen tubegrowth, the ntp303 5′ UTR increased the expression of the luciferasegene to a level that was almost 8-fold higher than the control 5′ UTR(FIG. 3 b). This enhancement effect was absent in the ntp303 3′ UTRconstruct. No significant differences in expression level of the controlUTRs and the ntp303 UTR containing constructs were observed duringpollen development (FIG. 3 a).

To exclude the possibility that the expression enhancement mediated bythe ntp303 5′ UTR in growing pollen tubes was the result of a specificinteraction between the 5′ UTR and the firefly luciferase coding region,the firefly luciferase coding region was replaced by the Renillaluciferase coding region in the constructs syn44 5′/35S 3′ and 3035′/303 3′. The firefly and the Renilla luciferase mRNAs exhibit nosignificant sequence identity with each other. Normalization of theexpression of these constructs was established by co-bombardment with aconstruct containing the syn44 5′ UTR, the 35S termination sequence andthe firefly luciferase coding region. As is shown in FIG. 4, the ntp303UTRs gave rise to an expression level that was approximately 7-foldhigher than that of the control UTRs. Since this enhancement effect ofthe ntp303 5′ UTR was also found for firefly luciferase mRNAs, thisexcludes a specific interaction between the ntp303 5′ UTR and the codingregion.

EXAMPLE 2

Translational Enhancement During Pollen Tube Growth

To investigate whether the enhancement mediated by the ntp303 5′ UTRduring pollen tube growth was either the result of apost-transcriptional regulation event or of a burst in transcription(i.e. transcriptional regulation), we determined the expression andtranscription levels of syn99 5′/35S 3′, 303 5′/35S 3′ and syn99 5′/3033′ (table 1). 10 μg of total RNA isolated from a pollen extract that wasalso used for the determination of the expression level as measured fromthe luciferase activity, was hybridized with a ³²P-labeled luciferaseprobe. The transcription level was determined by calculation of theratio of the hybridization signal of the firefly luciferase mRNA to theRenilla luciferase mRNA hybridization signal of the co-bombarded^(R)syn44 5′/35S 3′ construct. After 20 hours of pollen tube growth, thentp303 5′ UTR showed a relative transcription level that wasapproximately 2-fold higher than that of the syn99 5′ UTR. The constructcontaining the ntp303 5′ UTR increased the relative expression 50-foldas compared to syn99 5′/35S 3′. Thus, pollen tube growth chimericluciferase transcripts containing the ntp303 5′ UTR are translated moreefficiently than luciferase mRNAs containing the control 5′ UTR.

TABLE 1 Analysis of the relative transcription (represented as relativeluc mRNA abundance) and translation (represented as relative LUCactivity) levels of different UTR gene fusion constructs. See resultssection for a description of the followed methodology. The values inparentheses represents the relative transcription and translation levelsafter normalization to the relative values of the syn99 5′/35S 3′construct. Measurements were assayed after 20 hours of pollen tubegrowth. Relative luc mRNA Relative LUC Activity Construct Abundance(rlu/10 sec⁻¹) syn99 5′/35S 3′ 1.36 SE ± 0.17 (1.00) 1.02 SE ± 0.21(1.00) 303 5′/35S 3′ 3.70 SE ± 0.22 (2.72) 50.60 SE ± 0.22 (49.61) syn995′/35S 3′ 1.81 SE ± 0.22 (1.33) 2.50 SE ± 0.42 (2.45)

EXAMPLE 3

The 5′ UTR-Mediated Enhancement of Gene Expression also Occurs in OtherCells Hopes than Growing Pollen Tubes

To test whether the ntp303 5′ UTR mediated enhancement of expression ingrowing pollen tubes was restricted to a pollen-specific environment,the constructs syn44 5′/35S 3′ and 303 5′/303 3′ were reconstructed byreplacing their ntp303 promoter with the CaMV 35S promoter. The CaMV 35Spromoter is almost inactive in pollen tubes, but highly active insporophytic tissues. After particle bombardment of these constructs intomature pollen and young leaves followed by 20 hours of in vitroincubation, transient expression was assayed. Normalization of theexpression of these constructs was done with the expression level of aco-bombarded construct containing the CaMV 35S promoter, the syn44 5′UTR, the Renilla luciferase reporter gene and the 35S 3′ UTR. In growingpollen tubes, the ntp303 5′ UTR increased the transient expression to alevel that was approximately S-fold higher than the control UTRs (FIG. 5a). The differences in the expression level approached that of theconstructs containing the same UTR combinations but linked to the ntp303promoter (compare FIGS. 5 a and 2 b). In young leaves, the ntp303 5′ UTRincreased the expression to a level that was approximately 2-fold higherthan the control UTRs (FIG. 5 b). These data demonstrate that the ntp3035′ UTR-mediated enhancement of expression also occurs in other celltypes than growing pollen tubes, such as sporophytic cells. However, thentp303 5′ UTR-mediated enhancement of expression is highest in growingpollen tubes.

EXAMPLE 4

The Enhancement of Expression Dung Pollen Tube Growth is Attributable toSpecific Regions within the ntp303 5′ UTR

FIG. 6 illustrates the predicted secondary structure of the ntp303 5′UTR as analyzed with the RNAdraw software package (Hofacke et al.,(1994) Chem. Monthly 125, 167-188). There are two putative stem-loopstructures designated H-I (nucleotides 4-76 of SEQ ID No. 1) and H-II(nucleotides 104-151 of SEQ ID No. 1). The H-I stem-loop structure islocated at the 5′-terminus and has a calculated energy value (ΔG) of −64kJ/mol. This structure contains eight repeats of a GAA triplet(nucleotides 27-50 of SEQ ID No. 1) in the external loop and a doublestranded RNA in the stem of the predicted H-I structure, consisting ofGAAGAAGA (14-21) and the complementary strand TCTTCTTC (59-66). The H-IIstructure is located 22 nucleotides upstream from the translationinitiation site and has a calculated energy value (ΔG) of −26 kJ/mol.The effect of sequences that reside within the H-I and H-II structureson enhancement of expression during pollen tube growth was investigatedby a series of ntp303 5′ UTR deletion constructs (FIGS. 7 a and c).These constructs were bombarded into mature pollen and their expressionwas assayed after 20 hours of pollen tube growth (FIGS. 7 b and d). Analmost complete inactivation of reporter gene expression was achievedafter deletion of the last 70 nucleotides at the 3′ terminus of thentp303 5′ UTR which included the complete H-II structure (Δ70 303 5′/35S3′ (SEQ ID NO: 19)) (FIG. 7 d). The same was true after internaldeletion of only the H-II structure (ΔH-II 303 5′/35S 3′ (SEQ ID NO:20)) (FIG. 7 d). In both cases, the expression values were in the samerange as the background values (i.e. the measured autoluminescence ofthe luciferine substrate). FIG. 7 b shows the transient expression ofgene fusion constructs with deletions within the H-I stem-loopstructure. The lowest level of transient expression was found afterinternal deletion of the (GAA)₈ repeat (AGAA 303 5′/35S 3′ (SEQ ID NO:17)). This expression level was comparable with the expression level ofthe control construct containing the syn99 leader (data not shown). Adecrease in transient expression of approximately 94% occurred afterdeletion of the first 55 nucleotides (Δ55 303 5′/35S 3′ (SEQ ID NO: 16))at the 5′ terminus including the (GAA)₈ repeat. Deletion of the first 29nucleotides at the 5′ terminus of the ntp303 5′ UTR (Δ29 303 5′/35S 3′(SEQ ID NO: 15)) caused only a slight decrease in transient expressioncompared to that of the unmodified ntp303 5′ UTR. These results clearlydemonstrate that luciferase transcripts which contain deletions withinthe ntp303 5′ UTR are expressed to a lower level than transcriptscontaining the unmodified ntp303 5′ UTR. Deletions within or of the H-Ior H-II structure caused a different reduction in transient expression.Absence of the enhancement effect was observed after deletion of the(GAA)₈ repeat, whereas deletion of the complete H-II structure caused acomplete collapse in expression.

EXAMPLE 5

The H-1 and H-II Structures in the ntp303 5′UTR Influence theTranslation Efficiency Efficiency

Whether the decrease in transient expression of the 5′ UTR deletionconstructs was the result of a change in the transcription ortranslation efficiency, was investigated by measuring the relativetranscription and translation levels of some of the ntp303 5′ UTRdeletion constructs (table 2). The relative transcription levels of theconstructs containing deletions of the complete H-II structure (Δ70 3035′/35S 3′ and ΔH-II 303 5′/35S 3′) dropped to a level that was lowerthan that of syn99 5′/35S 3′. Internal deletion of the (GAA)₈ repeat(AGAA 303 5′/35S 3′) resulted in a relative transcription level that wassomewhat lower than the transcription level of the construct containingthe unmodified ntp303 5′ UTR, but the relative transcription levelsremained higher than that of the construct containing the synthetic 5′UTR. In contrast to the effects of either the deletion of the (GAA)₈repeat or the H-II structure on the relative transcription level, a moredrastic effect was observed for the relative translation levels. Adrastic decrease in the relative translation level was observed afterdeletion of the H-II structure, the values of the normalized translationlevel were in the range of the background values. Deletion of the (GAA)₈repeat revealed an almost 2-fold lower relative translation levelcompared to 303 5′/303 3′. From these data, we conclude that the drop inexpression observed after deletion of either the H-I -or H-II structuresis mainly the result of a decrease in the translation efficiency. Themost severe effect on the decrease in the translation efficiency wasfound after deletion of the H-II structure.

TABLE 2 Analysis of the relative transcription (represented as relativeluc mRNA abundance) and translation (represented as relative LUCactivity) levels of different ntp303 5′ UTR gene fusion constructs. Seeresults section for a description of the followed methodology. Thevalues in parentheses represents the relative transcription andtranslation 33 levels after normalization to the relative values of thesyn99 5′/35S 3′ construct. Measurements were assayed after 20 hours ofpollen tube growth. Relative luc mRNA Relative LUC Activity ConstructAbundance (counts) (rlu/10 sec⁻¹) 303 5′/35S 3′ 2.97 SE ± 0.09 (2.18)31.08 SE ± 5.61 (30.47) ΔAAG 303 5′/35S 3′ 2.30 SE ± 0.51 (1.69) 17.49SE ± 4.76 (17.15) Δ70 303 5′/35S 3′ 1.13 SE ± 0.06 (0.83) 0.01 SE ± 0.00(0.01) ΔH-II 303 5′/35S 3′ 0.82 SE ± 0.09 (0.60) 0.01 SE ± 0.00 (0.01)

1-21. (canceled)
 22. A nucleic acid construct comprising a firstnucleotide sequence that has at least 65% nucleotide sequence identitywith the nucleotide sequence of SEQ ID NO: 1, operably linked to asecond nucleotide sequence encoding a protein.
 23. The nucleic acidconstruct of claim 22, wherein said first nucleotide sequence comprisesa nucleotide sequence that consists of a repeat of 8 GAA units.
 24. Thenucleic acid construct of claim 22, wherein said first nucleotidesequence comprises a nucleotide sequence that has at least 90%nucleotide sequence identity to nucleotides 104-151 of the nucleotidesequence of SEQ ID NO:
 1. 25. The nucleic acid construct of claim 22,wherein said first nucleotide sequence comprises: (i) a nucleotidesequence that consists of a repeat of 8 GAA units; and, (ii) anucleotide sequence that has at least 90% nucleotide sequence identityto nucleotides 104-151 of the nucleotide sequence of SEQ ID NO:
 1. 26.The nucleic acid construct of claim 22, wherein the nucleotide sequenceencoding the protein is further operably linked to a promotor.
 27. Thenucleic acid construct of claim 22, wherein the first nucleotidesequence comprises the sequence of SEQ ID NO:
 1. 28. A recombinantanimal, mammalian, plant, fungal or bacterial host cell containing oneor more copies of the nucleic acid construct as defined by claim
 22. 29.A method for expressing a polypeptide of interest in a cell, wherein thecell is a plant, fungal, bacterial, animal or mammalian cell, comprisingthe steps of: a. providing a nucleic acid construct comprising: i) afirst nucleotide sequence having at least 65% nucleotide sequenceidentity to SEQ ID NO: 1 operably linked to ii) a second nucleotidesequence encoding a polypeptide of interest, and further operably linkedto iii) a promoter; and b. transforming the cell with said nucleic acidconstruct to obtain a transformed cell; and c. expressing thepolypeptide in said transformed cell.
 30. The method of claim 29,wherein said first nucleotide sequence comprises a nucleotide sequencethat consists of a repeat of 8 GAA units.
 31. The method of claim 29,wherein said first nucleotide sequence comprises a nucleotide sequencethat has at least 90% nucleotide sequence identity to nucleotides104-151 of the nucleotide sequence of SEQ ID NO:
 1. 32. The method ofclaim 29, wherein said first nucleotide sequence comprises: (i) anucleotide sequence that consists of a repeat of 8 GAA units; and, (ii)a nucleotide sequence that has at least 90% nucleotide sequence identityto nucleotides 104-151 of the nucleotide sequence of SEQ ID NO:
 1. 33.The method of claim 29, wherein said first nucleotide sequence comprisesthe sequence of SEQ ID NO:
 1. 34. The method of claim 29, wherein thepolypeptide of interest is heterologous to said cell.
 35. The method ofclaim 29, wherein the polypeptide is expressed in a cell of a doubledhaploid homozygous transgenic Nicotiana tabacum plant silenced forntp303.
 36. The method of claim 29, wherein the polypeptide is expressedin the pollen or seed of a plant that comprises said plant cell.
 37. Themethod of claim 29, wherein the promoter of (a)(iii) is heterologous tosaid second nucleotide sequence.
 38. The method of claim 29, wherein thefirst nucleotide sequence comprises a sequence forming an HI and HIIstructure.
 39. The method of claim 29, wherein the first nucleotidesequence is DNA.
 40. The method of claim 29, wherein the firstnucleotide sequence is RNA.
 41. The method of claim 29, furthercomprising step (d) recovering said polypeptide.